DETERMINANTS OF PERFORMANCE IN THE ISOCITRATE DEHYDROGENASE OF ESCHERICHIA-COLI

The substrate specificity of the NADP-dependent isocitrate dehydrogena se oi Escherichia coli was investigated by combining site-directed mut agenesis and utilization of alternative substrates. A comparison of th e kinetics of the wild-type enzyme with 2R-malate reveals that the gam ma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzyme s and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carbox ylate of bound isocitrate, a factor of 150 to the negative charge of t he gamma-carboxylate, and a factor of 50 for the gamma-methyl. These r esults are entirely consistent with X-ray structures of Michaelis comp lexes that show a hydrogen bond positions the gamma-carboxylate of iso citrate so that a salt bridge can form to the nicotinamide ring of NAD P.